Non-necrotic cellular death is controlled by a genetic program termed Program Cell Death or Apoptosis. When a cell receives a death signal, it turns on this program which leads to systematic degradation of macromolecules and structures and its eventual phagocytic elimination. In the absence of this program, a cell lives or survives (LIFE). It is, however, not clear whether there is a distinct genetic program for LIFE as in the case of death. At present, knowledge on cell LIFE and DEATH is scanty and a clear working hypothesis is lacking. Current concepts hold that the program for cell death is constitutive and cells survive only because survival regulatory proteins such as bcl-2, bcl-xL, A1 and Mcl1 inhibit the program. Death regulators such as bax, bak, bad, and bik are believed to accelerate the death program. However, something is not quite right with this hypothesis. From the Darwinian theory of evolution, we deduce that the primary goal of an organism is to survive so as to ensure the preservation of its species. It chooses to die only when natural selection no longer favors its genetic makeup. Knowing that a cell is the fundamental unit of the organism and that in some cases, the organism consists of just a cell, why then would cells create a reverse genetic program in which death is the primary purpose and survival is conditional? It would be more logical that cells have two mutually exclusive programs; one for life and the other for death. Survival regulators would sustain the LIFE program and DEATH regulators would activate the death program only when the life program fails. If a specific genetic program for LIFE exists, understanding it may fundamentally change the way we view life or devise medical treatment for diseases. A novel approach to test these concepts is to perturb the LIFE/DEATH process by enforced overexpression of bcl-2 (or any of the other survival regulators), and then analyze for genes with significant differential expression. Such genes could constitute indirect targets through which bcl-2 and other survival regulators enhance LIFE. Initial analysis using Differential RNA Display (DRD) technique identified a few gene fragments that were differentially expressed in response to bcl-2 overexpression. They are previously unknown genes since they lack significant homology to genes in nucleic acid databases. Because of the high degree of false positives associated with DRD method, and a subsequent observation that the results of Northern analysis did not confirm the high degree of differential expression obtained with DRD, a new method is currently being developed to reanalyze for differential expression in response to bcl-2 overexpression. The new method, Differential Expression Profiling by Expressed Sequence Tags (DEPEST), consists of creating ds cDNAs for mRNA from the test and control cells, using a 5'-biotynylated anchored oligo dT primer with a rare restriction site (Not 1). The cDNA libraries are digested to completion with a 4-base cutting restriction enzyme (Sau 3) to yield 256 bp fragments on the average. The 3'-prime-most fragments are purified using streptavidin-coated magnetic beads and ligated to form ditags. The ditags are then released by Not 1 digestion, cloned into Not 1 digested vector and sequenced. By using a specific software, the sequences of the test and control tags will be compared and statistically analyzed to identify those that are differentially expressed. Those that show high degree of differential expression will then be characterized for functional relevance in a putative genetic program for LIFE.